Hemostatic compositions

ABSTRACT

The invention discloses a hemostatic composition comprising crosslinked gelatin in particulate form suitable for use in hemostasis, wherein the composition is present in paste form containing 15.0 to 19.5% (w/w), preferably 16.0 to 19.5% (w/w), 16.5 to 19.5% (w/w), 17.0 to 18.5% (w/w) or 17.5 to 18.5% (w/w), more preferred 16.5 to 19.0% (w/w) or 16.8 to 17.8% (w/w), especially preferred 16.5 to 17.5% (w/w), and wherein the composition comprises an extrusion enhancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 61/552,270, filedOct. 27, 2011, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to hemostatic compositions and processesfor making such compositions.

BACKGROUND OF THE INVENTION

Hemostatic compositions in dry storage-stable form that comprisebiocompatible, biodegradable, dry stable granular material are knowne.g. from WO98/008550A or WO2003/007845A. These products have beensuccessfully applied on the art for hemostasis. Floseal® is an examplefor a highly effective haemostatic agent consisting of a granulargelatin matrix swollen in a thrombin-containing solution to form aflowable paste.

Since such products have to be applied to humans, it is necessary toprovide highest safety standards for quality, storage-stability andsterility of the final products and the components thereof. In addition,manufacturing and handling should be made as convenient and efficient aspossible.

A successful product in this field (the Floseal® product mentionedabove) utilizes a gelatin matrix used in conjugation with areconstituted lyophilized thrombin solution. The gelatin matrix isapplied as a flowable granular form of gelatin and thrombin with agelatin content of about 11 to 14.5%. Lower gelatin content results in arunny product with diminished performance due to difficulties in havingthe product remain at the treatment site, especially under conditions ofhigh blood flow. Higher gelatin particle concentration leads to aproduct that is difficult to deliver by usual means of administration,such as syringes or catheters, due to higher resistance to flow. Theinclusion of plasticizers in the composition, e.g., polyethyleneglycols, sorbitol, glycerol, and the like has been suggested(EP0927053B1) and can diminish extrusion force, but inclusion of thesematerials does not necessarily improve performance.

It is an object of the present invention to provide a hemostaticcomposition based on a crosslinked gelatin with improved adhering andhemostatic properties compared to the gelatin products such as Flosealaccording to the prior art and methods for making such hemostaticcompositions. The compositions should also be provided in a convenientand usable manner, namely as a flowable paste usable in endoscopicsurgery and microsurgery. The products must have an extrusion force of40 N or below, preferably below 35 N, especially preferred below 20 N.The products should preferably be provided in product formats enabling aconvenient provision of “ready-to-use” hemostatic compositions, whichcan be directly applied to an injury without any time consumingreconstitution steps.

SUMMARY OF THE INVENTION

Therefore, the present invention provides a hemostatic compositioncomprising crosslinked gelatin in particulate form suitable for use inhemostasis, wherein the composition is present in paste form containing15.0 to 19.5% (w/w) crosslinked gelatin, preferably 16.0 to 19.5% (w/w),16.5 to 19.5% (w/w), 17.0 to 18.5% (w/w) or 17.5 to 18.5% (w/w), morepreferred 16.5 to 19.0% (w/w) or 16.8 to 17.8% (w/w), especiallypreferred 16.5 to 17.5% (w/w), and wherein the composition comprises anextrusion enhancer, especially albumin.

The invention also refers to the use of this hemostatic composition fortreating an injury selected from the group consisting of a wound, ahemorrhage, damaged tissue and/or bleeding tissue comprisingadministering such a hemostatic composition and kits making such ahemostatic composition for the treatment of such injury.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention provides a hemostatic composition comprisingcrosslinked gelatin in particulate form suitable for use in hemostasis,wherein the composition is present in paste form containing 15.0 to19.5% (w/w) crosslinked gelatin (=weight of dry crosslinked gelatin perweight of final composition), preferably 16.0 to 19.5% (w/w), 16.5 to19.5% (w/w), 17.0 to 18.5% (w/w) or 17.5 to 18.5% (w/w), more preferred16.5 to 19.0% (w/w) or 16.8 to 17.8% (w/w), especially preferred 16.5 to17.5% (w/w), and wherein the composition comprises an extrusionenhancer.

It has been surprisingly found within the course of the presentinvention that the provision of extrusion enhancers, such as albumin inthe appropriate amount, enables the use of higher gelatin concentrationsand that the use of higher gelatin concentrations improves thehemostatic properties of such products. This is an effect which is notsuggested in the prior. Moreover, it was surprising that higherconcentration of crosslinked gelatin result in better adhesiveproperties (in contrast to the results known in the prior art (e.g. FIG.4 of WO2008/076407A2).

For enabling the preferred properties due to the higher gelatinconcentrations in the paste according to the present invention, it isnecessary to provide the extrusion enhancers in appropriate amounts. Theamounts shall be high enough so as to obtain the extrusion effect, i.e.to enable a flowable paste even for amounts of 15 to 19.5% crosslinkedgelatin so that the hemostatic composition can be applied e.g. inmicrosurgery; on the other hand, the amounts shall be as low as toprevent negative functional properties of the hemostatic composition,for example adherence to wounds or hemostatic performance. For example,if the extrusion enhancer is albumin (which is specifically preferred,especially human serum albumin), it must be provided in an amount ofbetween 0.5 to 5.0% (w/w) (=weight of extrusion enhancer per weight offinal composition), preferably 1.0 to 5.0% (w/w), preferably 2.0 to 4.5%(w/w), more preferred 1.5 to 5.0% (w/w), especially preferred about 1.5%(w/w).

Another preferred class of extrusion enhancers according to the presentinvention are phospholipids, such as phosphatidylcholine and -serine, orcomplex mixtures such as lecithins or soy bean oils.

In another preferred embodiment the present invention provides ahemostatic composition comprising crosslinked gelatin in particulateform suitable for use in hemostasis, wherein the composition is presentin paste form containing 16.0 to 19.5% (w/w), preferably 16.5 to 19.5%(w/w 17.0 to 18.5% (w/w) or 17.5 to 18.5% (w/w), more preferred 16.5 to19.0% (w/w) or 16.8 to 17.8% (w/w), especially preferred 16.5 to 17.5%(w/w), and wherein the composition comprises an extrusion enhancer.Preferably the extrusion enhancer is human serum albumin.

In another preferred embodiment the present invention provides ahemostatic composition comprising crosslinked gelatin in particulateform suitable for use in hemostasis, wherein the composition is presentin paste form containing 15.0 to 19.5% (w/w) crosslinked gelatin,preferably 16.0 to 19.5% (w/w), 16.5 to 19.5% (w/w), 17.0 to 18.5% (w/w)or 17.5 to 18.5% (w/w), more preferred 16.5 to 19.0% (w/w) or 16.8 to17.8% (w/w), especially 16.5 to 17.5% (w/w), and wherein the compositioncomprises an extrusion enhancer in a concentration of more than 0,8%(w/w), preferably about 3,3% (w/w). Preferably the extrusion enhancer ishuman serum albumin, e.g. in the above mentioned concentrations.

The hemostatic compositions according to the present invention,especially the ones that use albumin as extrusion enhancer, havespecific advantages over the compositions using lower amounts ofcrosslinked gelatin (13 to 14.5%), especially they have an enhanced invivo efficacy. It was unexpectedly revealed within the course of thepresent invention that a formulation with a higher gelatin particleconcentration results in greater hemostatic performance both in ex vivotest methods that use whole human blood and in pre-clinical animalexperiments. The products according to the present invention enable areduced surgical approximation time and a faster time to hemostasis.

The compositions according to the present invention have a meanextrusion force (employing the test method described in example 1) of 40N or below, preferably below 35 N, especially preferred below 20 N.

According to preferred embodiment of the present invention, thehemostatic composition comprises glutaraldehyde-crosslinked gelatin orgenipin(Methyl(1R,2R,6S)-2-hydroxy-9-(hydroxymethyl)-3-oxabicyclo[4.3.0]nona-4,8-diene-5-carboxylate)-crosslinkedgelatin, preferably type B gelatin, more preferably type B gelatin ofhide origin.

Preferably, the crosslinked gelatin is present as granular material.

The hemostatic composition according to the present invention preferablycomprises a gelatin polymer which is especially a type B gelatinpolymer. Type B gelatin has proven to be specifically advantageous foruse in hemostatic agents as the base treatment is highly effective ingenerating gelatin of appropriate properties and in mitigating risk ofviral and zoonotic infection. A specifically preferred gelatinpreparation can be prepared by processing young bovine corium with 2 NNaOH for about 1 hour at room temperature, neutralizing to pH 7-8, andheating to 70° C. The corium is then fully solubilized to gelatin with3-10% (w/w), preferably 7-10% (w/w) gelatin in solution. This solutioncan be cast, dried and ground to provide gelatin type B powder.

Preferably, the gelatin has a Bloom strength of 200 to 400, especially atype B gelatin with a Bloom strength of 200 to 400. Bloom is a test tomeasure the strength of gelatin. The test determines the weight (ingrams) needed by a probe (normally with a diameter of 0.5 inch) todeflect the surface of the gel 4 mm without breaking it. The result isexpressed in Bloom (grades). To perform the Bloom test on gelatin, a6.67% gelatin solution is kept for 17-18 hours at 10° C. prior to beingtested.

The hemostatic composition according to the present invention preferablycontains the crosslinked gelatin in particulate form, especially asgranular material. This granular material can rapidly swell when exposedto a fluid (i.e. the diluent) and in this swollen form is capable ofcontributing to a flowable paste that can be applied to a bleeding site.According to a preferred embodiment, the crosslinked gelatin is providedfrom a dry crosslinked gelatin. This dry crosslinked gelatin powder canbe prepared to re-hydrate rapidly if contacted with a pharmaceuticallyacceptable diluent. The gelatin granules, especially in the form of agelatin powder, preferably comprise relatively large particles, alsoreferred to as fragments or sub-units, as described in WO98/08550A andWO2003/007845A. A preferred (median) particle size will be the rangefrom 10 to 1.000 μm, preferably from 200 to 800 μm, but particle sizesoutside of this preferred range may find use in many circumstances.

Usually, the gelatin particles have a mean particle diameter (“meanparticle diameter” is the median size as measured by laserdiffractometry; “median size” (or mass median particle diameter) is theparticle diameter that divides the frequency distribution in half; fiftypercent of the particles of a given preparation have a larger diameter,and fifty percent of the particles have a smaller diameter) from 10 to1000 μm, preferably 50 to 700 μm, 200 to 700 μm, 300 to 550 μm,especially preferred 350 to 550 μm (median size). Although the termspowder and granular (or granulates) are sometimes used to distinguishseparate classes of material, powders are defined herein as a specialsub-class of granular materials. In particular, powders refer to thosegranular materials that have the finer grain sizes, and that thereforehave a greater tendency to form clumps when flowing. Granules includecoarser granular materials that do not tend to form clumps except whenwet.

The present crosslinked gelatin in particulate form suitable for use inhemostasis may include dimensionally isotropic or non-isotropic forms.For example, the crosslinked gelatin in the kit according to the presentinvention may be granules or fibers; and may be present in discontinuousstructures, for example in powder forms.

The dry gelatin composition is liquid absorbing. For example, uponcontact with liquids, e.g. aqueous solutions or suspensions (especiallya buffer or blood) the crosslinked gelatin takes up the liquid and willdisplay a degree of swelling, depending on the extent of hydration. Thematerial preferably absorbs from at least 400%, preferably about 500% toabout 2000%, especially from about 500% to about 1300% water or aqueousbuffer by weight, corresponding to a nominal increase in diameter orwidth of an individual particle of subunit in the range from e.g.approximately 50% to approximately 500%, usually from approximately 50%to approximately 250%. For example, if the (dry) granular particles havea preferred size range of 0.01 mm to 1.5 mm, especially of 0.05 mm to 1mm, the fully hydrated composition (e.g. after administration on a woundor after contact with an aqueous buffer solution) may have a size rangeof 0.05 mm to 3 mm, especially of 0.25 mm to 1.5 mm.

The dry compositions will also display a significant “equilibrium swell”when exposed to an aqueous re-hydrating medium (=pharmaceuticallyacceptable diluent, also referred to as reconstitution medium).Preferably, the swell will be in the range from 400% to 1300%,preferably 400% to 1000%, more preferred 500% to 1100%, especiallypreferred from 500% to 900%, depending on its intended use. Suchequilibrium swell may be controlled e.g. (for a crosslinked polymer) byvarying the degree of cross-linking, which in turn is achieved byvarying the cross-linking conditions, such as the duration of exposureof a cross-linking agent, concentration of a cross-linking agent,cross-linking temperature, and the like. Materials having differingequilibrium swell values perform differently in different applications.The ability to control crosslinking and equilibrium swell allows thecompositions of the present invention to be optimized for a variety ofuses. In addition to equilibrium swell, it is also important to controlthe hydration of the material immediately prior to delivery to a targetsite. Hydration and equilibrium swell are, of course, intimatelyconnected. A material with 0% hydration will be non-swollen. A materialwith 100% hydration will be at its equilibrium water content. Hydrationsbetween 0% and 100% will correspond to swelling between the minimum andmaximum amounts. “Equilibrium swell” may be determined by subtractingthe dry weight of the gelatin hydrogel powder from its weight when fullyhydrated and thus fully swelled. The difference is then divided by thedry weight and multiplied by 100 to give the measure of swelling. Thedry weight should be measured after exposure of the material to anelevated temperature for a time sufficient to remove substantially allresidual moisture, e.g., two hours at 120° C. The equilibrium hydrationof the material can be achieved by immersing the dry material in apharmaceutically acceptable diluent, such as aqueous saline, for a timeperiod sufficient for the water content to become constant, typicallyfor from 18 to 24 hours at room temperature.

The crosslinked gelatin may be provided as a film which can then bemilled to form a granular material. Most of the particles contained in agranular material (e.g. more than 90% w/w) have preferably particlesizes of 10 to 1.000 μm, preferably 50 to 700 μm, 200 to 700 μm, 300 to550 μm, especially preferred 350 to 550 μm.

Preferably, the flowable form of the hemostatic composition containsparticles that are more than 50% (w/w) with a size of 100 to 1000 μm,preferably more than 80% (w/w) with a size of 100 to 1000 μm.

Examples of suitable gelatin materials for crosslinking are describedi.a. in examples 1 and 2 of EP1803417B1 and example 14 of U.S. Pat. No.6,066,325A and U.S. Pat. No. 6,063,061A. Gelatin may also be used withprocessing aids, such as PVP, PEG and/or dextran as re-hydration aids.

In one particular aspect of the present invention, compositions willcomprise crosslinked gelatin powders having a moisture content of 20%(w/w) or less, wherein the powder was crosslinked in the presence of are-hydration aid so that the powder has an aqueous re-hydration ratewhich is at least 5% higher than the re-hydration rate of a similarpowder prepared without the re-hydration aid. The “re-hydration rate” isdefined according to EP1803417B1 to mean the quantity of an aqueoussolution, typically 0.9% (w/w) saline that is absorbed by a gram of thepowder (dry weight basis) within thirty seconds, expressed as g/g. Therehydration rate is measured by mixing the crosslinked gelatin withsaline solution for 30 seconds and depositing the wet gelatin on afilter membrane under vacuum to remove the free aqueous solution. Onethen records the weight of the wet gelatin retained on the filter, driesit (e.g. 2 hr at 120° C.), then records the dry weight of the gelatinand calculates the weight of solution that was absorbed per gram of drygelatin.

Preferred compositions of the present invention will have a re-hydrationrate of at least 3 g/g, preferably at least 3.5 g/g, and often 3.75 g/gor higher. Re-hydration rates of similar powders prepared without there-hydration aids are typically below three, and a percentage increasein re-hydration rate will usually be at least 5%, preferably being atleast 10%, and more preferably being at least 25% or higher.

Crosslinking can be done with any suitable crosslinker, e.g.glutaraldehyde such as e.g. described in WO98/08550A and WO2003/007845A.Crosslinking can also be carried out with a non-toxic crosslinker suchas genipin and the like.

Production cost is less for a genipin crosslinked gelatin productaccording to the present invention than a glutaraldehyde crosslinkedone, since reagent, energy, and time costs are lower. The genipincrosslinked gelatin reaction can be performed in water at neutral pH atroom temperature for ≦16 hours. The product can be cleaned-up by anethanol and/or water wash which is not only cheaper, but moreimportantly, safer for the operator.

The method preferably applies the gelatin as being present in dry formbefore the crosslinking step.

The preferred genipin-type crosslinker according to the presentinvention is, of course, genipin(Methyl(1R,2R,6S)-2-hydroxy-9-(hydroxymethyl)-3-oxabicyclo[4.3.0]nona-4,8-diene-5-carboxylate);however, also other crosslinkers of the iridoid- or secoiridoid-type maybe used, such as oleuropein. Preferred concentrations of genipin forcrosslinking are in the range of 0.5 to 20 mM, preferably 1 to 15 mM,especially 2 to 10 mM.

According to a preferred embodiment of the present invention, thegenipin crosslinked gelatin is subjected to a quenching/oxidation stepwith oxidizing agents such as bleach, tBu-hydroperoxide, etc.,preferably to a treatment with sodium percarbonate, sodium hypochlorite,chlorine water or hydrogen peroxide (H₂O₂), especially preferred is atreatment with sodium percarbonate or H₂O₂ most preferred is a treatmentwith percarbonate.

Preferred H₂O₂ concentrations are 0.5 to 20% (w/w), especially 1 to 15%(w/w), more preferred about 5% (w/w). In an especially preferredembodiment the genipin concentration is between 5 to 10 mM, the reactiontime of gelatin with genipin is between 3 to 10 hours, especially 6hours, the H₂O₂ concentration is between 3 to 5% (w/w) and the reactiontime of the genipin-crosslinked gelatin with H₂O₂ is about 20 hours,

Preferred percarbonate concentrations are between 1 to 10% (w/w),especially 1 to 5% (w/w), more preferred 1 to 4% (w/w). In an especiallypreferred embodiment the genipin concentration is between 5 to 10 mM(especially about 8 nM), the reaction time of gelatin with genipin isbetween 3 to 10 hours (especially about 5 hours), the percarbonateconcentration is between 1 to 10% (w/w), especially preferred between 1to 4% w/w, and the reaction time of the genipin-crosslinked gelatin withpercarbonate is between 1 to 20 hours, preferably between 1 to 5 hours(e.g. 1, 2 or 3 hours).

Quenching may also be carried out in presence of antioxidants such assodium ascorbate or by controlling oxidation potential of the reactionenvironment such as carrying out quenching and/or genipin reaction in aninert atmosphere such as nitrogen or argon.

Preferred crosslinking reaction conditions include the performance inaqueous solution, preferably in a phosphate buffered saline(PBS)/ethanol buffer, especially at a pH of 4 to 12, preferably of 5.0to 10.0, especially of 6 to 8, or in deionized water or other aqueousbuffers which may contain between 0 to 50% of a water miscible organicsolvent. A PBS buffer contains physiological amounts of NaCl and KCl ina phosphate buffer at a physiological pH. An example for a PBS buffercontains 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄.2H₂O, 1.76 mM KH₂PO₄(pH=7.4). Another example of a PBS buffer consists of 137 mM NaCl, 2.7mM KCl, 4.3 mM Na₂HPO₄ and 1.4 mM KH₂PO₄ (pH=7.5).

The reaction may also be carried out in an aqueous buffer containing upto 50% of a water-miscible organic solvent and/or processing aids suchas PEG, PVP, mannitol, sodium percarbonate, sodium lactate, sodiumcitrate, sodium ascorbate etc.

Preferably, the crosslinking step is performed at a temperature of 4° C.to 45° C., preferably of 15 to 45° C., especially of 20 to 40° C.

The crosslinking step may be followed by a quenching step, especiallywith an amino-group containing quencher, preferably an amino acid,especially glycine. With the quencher, yet unreacted genipin-typecrosslinkers are inactivated (e.g. by reaction with the quencher inexcess) to prevent further crosslinking. Quenching may also be carriedout by raising pH of solution to between 8 to 14, or by usingnucleophilic compounds containing amino, thiol, or hydroxyl groups andalso a combination of pH raising and using nucelophilic compounds. Thequenching step after the genipin-gelatin crosslinking reaction accordingto the present invention can be actively directed to impart desiredphysical performance such as swell and TEG which are importantdeterminants of hemostatic activity above and beyond the generalgenipin-crosslinking alone.

The crosslinked gelatin is preferably washed after the crosslinkingstep, preferably by methanol, ethanol or water, especially by deionizedwater. Another preferred washing step applies an aqueous buffercontaining up to 50% (v/v) of water-miscible organic solvent and/or oneor more processing aids.

According to a preferred embodiment, the crosslinked gelatin is dried.In such a dried state, the hemostatic composition is storage-stable forlong time even at elevated temperatures (e.g. more than 20° C., morethan 30° C. or even more than 40° C.). Preferred dryness conditionsinclude crosslinked biocompatible polymers which are dried to have amoisture content of below 15% (w/w), preferably below 10%, morepreferred below 5%, especially below 1%. In another preferred embodimentthe product may be supplied in a hydrated or wet state where thehydrating solution may be a biocompatible buffer or solution.

A Glu-Gel product has a tendency to be camouflaged by surroundingtissue, since it's slightly yellow color blends in with it. This makesvisual evaluation of the desired application problematic. The genipincrosslinked gelatin products according to the present invention appearvariable color from pale yellow to dark blue or green based upon degreeof crosslinking reaction conditions, and subsequent processing andfinishing steps. This tunability of color and ability to obtain desiredcolor in finished product color has the added advantage of providingphysicians visual indication of proper product application in woundsites, since this color differentiates it from surrounding tissue,instead of potentially being camouflaged by it. This is another novelfeature of this invention. On the other hand, the color can be removedto obtain a non-colored product, depending on the needs with respect tothe final products.

In a preferred embodiment the biocompatible polymer, e.g. gelatin,crosslinked with a genipin-type crosslinker, e.g. genipin, is ahomogeneously (uniformely) crosslinked polymer as can be shown e.g. byfluorescence measurements as described in Example 3 of the presentapplication. In an especially preferred embodiment the biocompatiblepolymer, such as gelatin, is present as a homogeneously genipincrosslinked biocompatible polymer, such as gelatin, in particulate form.

A hemostatic composition according to the present invention ispreferred, wherein excipients, such as lubricants, e.g. hyaluronic acid,are present.

In another embodiment of the present invention excipients, such aslubricants, e.g. hyaluronic acid, are excluded.

The pharmaceutically acceptable diluent is preferably an aqueoussolution and may contain a substance selected from the group consistingof NaCl, CaCl₂ and sodium acetate. For example, a pharmaceuticallyacceptable diluent comprises water for injection, and—independently ofeach other—50 to 200 mM NaCl (preferably 150 mM), 10 to 80 mM CaCl₂(preferably 40 mM) and 1 to 50 mM sodium acetate (preferably 20 mM). Inanother embodiment the pharmaceutically acceptable diluent contains lessthan 35 g/l of mannitol, preferably less than 25 g/l, more preferredless than 10 g/l, especially preferred the pharmaceutically acceptablediluent is essentially free of mannitol.

According to a preferred embodiment, the pharmaceutically acceptablediluent comprises thrombin, preferably 10 to 1000 I.U. thrombin/ml,especially 250 to 700 I.U. thrombin/ml. Preferably, the hemostaticcomposition in this ready to use form contains 10 to 100.000International Units (I.U.) of thrombin, more preferred 100 to 10.000I.U., especially 500 to 5.000 I.U. Thrombin (or any other coagulationinducing agent, such as snake venom, a platelet activator, a thrombinreceptor activating peptide and a fibrinogen precipitating agent) can bederived from any thrombin preparation which is suitable for use inhumans (i.e. pharmaceutically acceptable). Suitable sources of thrombininclude human and bovine blood, plasma or serum (thrombin of otheranimal sources can be applied if no adverse immune reactions areanticipated), thrombin of recombinant origin (e.g. human recombinantthrombin) and autologous human thrombin can be preferred for someapplications.

The pharmaceutically acceptable diluent is used in an amount to achievethe desired end-concentration in the ready-to-use composition. Thethrombin preparation may contain other useful component, such as ions,buffers, excipients, stabilizers, etc. Preferably, the thrombinpreparation contains human albumin as the extrusion enhancer. Preferredsalts are NaCl and/or CaCl₂, both used in the usual amounts andconcentrations applied for thrombin (e.g. 0.5 to 1.5% NaCl (e.g. 0.9%)and/or 20 to 80 mM CaCl₂ (e.g. 40 mM)).

In a further embodiment, the diluent can also include a buffer or buffersystem so as to buffer the pH of the reconstituted dry composition,preferably at a pH of 3.0 to 10.0, more preferred of 6.4 to 7.5,especially at a pH of 6.9 to 7.1.

Establishment of appropriate amounts of crosslinked gelatin, diluent andextrusion enhancer may be made in the kit according to theaforementioned prerequisites: For example a) a vial with 0.736 to 0.995g dry crosslinked gelatin (corresponding to 15.0 to 19.5% (w/w) in thefinal product) may be provided and b) a second vial with 4 ml diluentwith 60 to 240 mg albumin and, optionally, thrombin at a concentrationof 500 I.U./ml and/or 40 mM CaCl₂. Alternatively, albumin may be addedin lyophilized form to the dry gelatin component a) of the kit. Forexample, a) a vial with 0.573 to 0.775 g dry crosslinked gelatin(corresponding to 15.0 to 19.5% (w/w) in the final product) thereof 48to 192 mg albumin may be provided and b) a second vial with 3.2 mldiluent and, optionally, thrombin at a concentration of 500 I.U./mland/or 40 mM CaCl₂.

The crosslinked gelatin component of the kit according to the presentinvention is preferably provided as a dry composition, wherein thecrosslinked gelatin is present in dry form.

A substantially dry crosslinked gelatin composition according to thepresent invention has a residual content of moisture which mayapproximately correspond to the moisture content of comparable availableproducts, such as Floseal® (Floseal, for example, has approximately8-12% moisture as a dry product).

The dry crosslinked gelatin in particulate form suitable for use inhemostasis in the kit according to the present invention is preferablygelatin in powder form, especially wherein the powder particles have amedian particle size of 10 to 1000 μm, preferably 50 to 700 μm, 200 to700 μm, 300 to 550 μm, especially preferred 350 to 550 μm. A “drygranular preparation of crosslinked gelatin” according to the presentinvention is in principle known e.g. from WO98/08550A. Preferably, thecrosslinked gelatin is a biocompatible, biodegradable dry stablegranular material.

According to another aspect, the present invention relates to ahemostatic composition according to the present invention for use in thetreatment of an injury selected from the group consisting of a wound, ahemorrhage, damaged tissue, bleeding tissue and/or bone defects.

Another aspect of the present invention is a method of treating aninjury selected from the group consisting of a wound, a hemorrhage,damaged tissue and/or bleeding tissue comprising administering ahemostatic composition according to the present invention to the site ofinjury.

According to another aspect, the present invention also provides amethod for delivering a hemostatic composition according to theinvention to a target site in a patient's body, said method comprisingdelivering a hemostatic composition produced by the process according tothe present invention to the target site. Although also the drycomposition can be directly applied to the target site (and, optionallybe contacted with a diluent at the target site, if necessary), it ispreferred to contact the dry hemostatic composition with apharmaceutically acceptable diluent before administration to the targetsite, so as to obtain a hemostatic composition according to the presentinvention in paste form.

In such a method, a kit for making a flowable paste of crosslinkedgelatin for the treatment of an injury selected from the groupconsisting of a wound, a hemorrhage, damaged tissue and/or bleedingtissue, may be applied, this kit comprising

-   a) a dry hemostatic composition comprising crosslinked gelatin in    particulate form to be reconstituted to a flowable paste containing    15.0 to 19.5% (w/w) crosslinked gelatin (=weight of dry gelatin to    weight of final composition), preferably 16.0 to 19.5% (w/w), 16.5    to 19.5% (w/w), 17.0 to 18.5% (w/w) or 17.5 to 18.5% (w/w), more    preferred 16.5 to 19.0% (w/w) or 16.8 to 17.8% (w/w), especially    preferred 16.5 to 17.5% (w/w), crosslinked gelatin and-   b) a pharmaceutically acceptable diluent for reconstitution of the    hemostatic composition, wherein either the composition or the    diluent comprises an extrusion enhancer, especially albumin, in a    suitable amount, for example (for albumin) in an amount which leads    to an albumin concentration in the reconstituted paste of between    0.5 to 5.0% (w/w) (=weight of extrusion enhancer per weight of final    composition), preferably 1.0 to 5.0% (w/w), preferably 2.0 to 4.5%    (w/w), more preferred 1.5 to 5.0% (w/w), especially preferred about    1.5% (w/w).

A preferred further component of such a kit is—specifically if thehemostatic composition is contained in dry form—a diluent forreconstitution (=re-hydration medium) of the hemostatic composition.Further components of the kit may be administration means, such assyringes, catheters, brushes, etc. (if the compositions are not alreadyprovided in the administration means) or other components necessary foruse in medical (surgical) practice, such as substitute needles orcatheters, extra vials or further wound cover means. Preferably, the kitaccording to the present invention comprises a syringe housing the dryand stable hemostatic composition and a syringe containing the diluent(or provided to take up the diluent from another diluent container).

In a preferred embodiment, the pharmaceutically acceptable diluent isprovided in a separate container. This can preferably be a syringe. Thediluent in the syringe can then easily be applied to the final containerfor reconstitution of the dry hemostatic compositions according to thepresent invention. If the final container is also a syringe, bothsyringes can be finished together in a pack. It is therefore preferredto provide the dry hemostatic compositions according to the presentinvention in a syringe which is finished with a diluent syringe with apharmaceutically acceptable diluent for reconstituting said dry andstable hemostatic composition.

According to a preferred embodiment, the final container furthercontains an amount of a stabilizer effective to inhibit modification ofthe polymer when exposed to the sterilizing radiation, preferablyascorbic acid, sodium ascorbate, other salts of ascorbic acid, or anantioxidant.

With such a pharmaceutically acceptable diluent, a ready to use form ofthe present hemostatic composition may be provided which can then bedirectly applied to the patient. Accordingly, also method for providinga ready to use form of a hemostatic composition according to the presentinvention is provided, wherein the hemostatic composition is provided ina first syringe and a diluent for reconstitution is provided in a secondsyringe, the first and the second syringe are connected to each other,and the fluid is brought into the first syringe to produce a flowableform of the hemostatic composition; and optionally returning theflowable form of the hemostatic composition to the second syringe atleast once. Preferably, the ready-to use preparations are present orprovided as hydrogels. Products of this kind are known in principle inthe art, yet in a different format. Therefore, a method for providing aready to use form of a hemostatic composition according to the presentinvention, wherein the hemostatic composition is provided in a firstsyringe and a diluent for reconstitution is provided in a secondsyringe, the first and the second syringe are connected to each other,and the diluent is brought into the first syringe to produce a flowableform of the hemostatic composition; and optionally returning theflowable form of the hemostatic composition to the second syringe atleast once, is a preferred embodiment of the present invention. Thisprocess (also referred to as “swooshing”) provides a suitableready-to-use form of the compositions according to the present inventionwhich can easily and efficiently be made also within short times, e.g.in emergency situations during surgery. This flowable form of thehemostatic composition provided by such a method is specificallysuitable for use in the treatment of an injury selected from the groupconsisting of a wound, a hemorrhage, damaged tissue, bleeding tissueand/or bone defects.

For stability reasons, such products (as well as the products accordingto the present invention) are usually provided in a dry form in a finalcontainer and brought into the ready-to-use form (which is usually inthe form of a (hydro)gel, suspension or solution) immediately beforeuse, necessitating the addition of a pharmaceutically acceptablediluents (=re-hydration medium).

According to another aspect, the present invention relates to a methodfor providing a ready to use form of a hemostatic composition accordingto the present invention, wherein the hemostatic composition is providedin a first syringe and a diluent for reconstitution is provided in asecond syringe, the first and the second syringe are connected to eachother, and the fluid is brought into the first syringe to produce aflowable form of the hemostatic composition; and optionally returningthe flowable form of the hemostatic composition to the second syringe atleast once.

Preferably, the flowable form of the hemostatic composition according tothe present invention contains more than 50% (w/w) particles with a sizeof 100 to 1000 μm, preferably more than 80% (w/w) particles with a sizeof 100 to 1000 μm.

The biocompatible hemostatic crosslinked polymer according to thepresent invention—once applied to a wound—forms an efficient matrixwhich can form a barrier for blood flow. Specifically the swellingproperties of the hemostatic polymer can make it an effective mechanicalbarrier against bleeding and re-bleeding processes.

The present composition may additionally contain a hydrophilic polymericcomponent (also referred to as “reactive hydrophilic component” or“hydrophilic (polymeric) crosslinker”) which further enhances theadhesive properties of the present composition. This hydrophilicpolymeric component of the haemostatic composition according to thepresent invention acts as a hydrophilic crosslinker which is able toreact with its reactive groups once the haemostatic composition isapplied to a patient (e.g. to a wound of a patient or another placewhere the patient is in need of a hemostatic activity). Therefore it isimportant for the present invention that the reactive groups of thehydrophilic polymeric component are reactive when applied to thepatient. It is therefore necessary to manufacture the haemostaticcomposition according to the present invention so that the reactivegroups of the polymeric component which should react once they areapplied to a wound are retained during the manufacturing process.

For hydrophilic polymeric crosslinkers whose reactive groups arehydrolysable, premature contact with water or aqueous liquids has to beprevented before administration of the haemostatic composition to thepatient, especially during manufacture. However, processing of thehydrophilic polymeric component during manufacturing may be possiblealso in an aqueous medium at conditions where the reactions of thereactive groups are inhibited (e.g. at a low pH). If the hydrophilicpolymeric components can be melted, the melted hydrophilic polymericcomponents can be sprayed or printed onto the matrix of crosslinkedgelatin. It is also possible to mix a dry form (e.g. a powder) of thehydrophilic polymeric component with a dry form of the crosslinkedgelatin. If necessary, then an increase of the temperature can beapplied to melt the sprinkled hydrophilic polymeric component to thecrosslinked gelatin to achieve a permanent coating of the haemostaticcomposition. Alternatively, these hydrophilic polymeric components canbe taken up into inert organic solvents (inert vis-à-vis the reactivegroups of the hydrophilic polymeric components) and brought onto thematrix of the crosslinked gelatin. Examples of such organic solvents aredry ethanol, dry acetone or dry dichloromethane (which are e.g. inertfor hydrophilic polymeric components, such as NHS-ester substitutedPEGs). Alternatively, nucleophilic groups may also be added (e.g.PEG-SH).

In a preferred embodiment the hydrophilic polymer component is a singlehydrophilic polymer component and is a polyalkylene oxide polymer,preferably a PEG comprising polymer. The reactive groups of thisreactive polymer are preferably electrophilic groups.

The reactive hydrophilic component may be a multi-electrophilicpolyalkylene oxide polymer, e.g. a multi-electrophilic PEG. The reactivehydrophilic component can include two or more electrophilic groups,preferably a PEG comprising two or more reactive groups selected fromsuccinimidylesters (—CON(COCH₂)₂), aldehydes (—CHO) and isocyanates(—N═C═O), e.g. a component as disclosed in the WO2008/016983 A(incorporated herein by reference in its entirety).

Preferred electrophilic groups of the hydrophilic polymeric crosslinkeraccording to the present invention are groups reactive to the amino-,carboxy-, thiol- and hydroxy-groups of proteins, or mixtures thereof.

Preferred amino group-specific reactive groups are NHS-ester groups,imidoester groups, aldehyde-groups, carboxy-groups in the presence ofcarbodiimides, isocyanates, or THPP (beta-[Tris(hydroxymethyl)phosphino]propionic acid), especially preferred isPentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate(=Pentaerythritoltetrakis[1-1′-oxo-5′-succinimidylpentanoate-2-poly-oxoethyleneglycole]ether(=an NHS-PEG with MW 10,000).

Preferred carboxy-group specific reactive groups are amino-groups in thepresence of carbodiimides.

Preferred thiol group-specific reactive groups are maleimides orhaloacetyls.

Preferred hydroxy group-specific reactive group is the isocyanate group.The reactive groups on the hydrophilic cross-linker may be identical(homo-functional) or different (hetero-functional). The hydrophilicpolymeric component can have two reactive groups (homo-bifunctional orhetero-bifunctional) or more (homo/hetero-trifunctional or more).

In special embodiments the material is a synthetic polymer, preferablycomprising PEG. The polymer can be a derivative of PEG comprising activeside groups suitable for cross-linking and adherence to a tissue.

By the reactive groups the hydrophilic reactive polymer has the abilityto cross-link blood proteins and also tissue surface proteins.Cross-linking to the crosslinked gelatin is also possible.

The multi-electrophilic polyalkylene oxide may include two or moresuccinimidyl groups. The multi-electrophilic polyalkylene oxide mayinclude two or more maleimidyl groups.

Preferably, the multi-electrophilic polyalkylene oxide is a polyethyleneglycol or a derivative thereof.

In a most preferred embodiment the hydrophilic polymeric component ispentaerythritolpoly(ethyleneglycol)ether tetrasuccinimidyl glutarate(═COH102, also pentaerythritoltetrakis[1-1′-oxo-5′-succinimidylpentanoate-2-poly-oxoethyleneglycole]ether).

The hydrophilic polymeric component is a hydrophilic crosslinker.According to a preferred embodiment, this crosslinker has more than tworeactive groups for crosslinking (“arms”), for example three, four,five, six, seven, eight, or more arms with reactive groups forcrosslinking. For example, NHS-PEG-NHS is an effective hydrophiliccrosslinker according to the present invention. However, for someembodiments, a 4-arm polymer (e.g. 4-arms-p-NP-PEG) may be morepreferred; based on the same rationale, an 8-arm polymer (e.g.8-arms-NHS-PEG) may even be more preferred for those embodiments wheremulti-reactive crosslinking is beneficial. Moreover, the hydrophiliccrosslinker is a polymer, i.e. a large molecule (macromolecule) composedof repeating structural units which are typically connected by covalentchemical bonds. The hydrophilic polymer component should have amolecular weight of at least 1000 Da (to properly serve as crosslinkerin the hemostatic composition according to the present invention);preferably the crosslinking polymers according to the present inventionhas a molecular weight of at least 5000 Da, especially of at least 8000Da.

For some hydrophilic crosslinkers, the presence of basic reactionconditions (e.g. at the administration site) is preferred or necessaryfor functional performance (e.g. for a faster cross-linking reaction atthe administration site). For example, carbonate or bicarbonate ions(e.g. as a buffer with a pH of 7.6 or above, preferably of 8.0 or above,especially of 8.3 and above) may be additionally provided at the site ofadministration (e.g. as a buffer solution or as a fabric or pad soakedwith such a buffer), so as to allow an improved performance of thehemostatic composition according to the present invention or to allowefficient use as a hemostatic and/or wound adherent material.

The reactivity of the hydrophilic polymeric component (which, asmentioned, acts as a crosslinker) in the composition according to thepresent invention is retained in the composition. This means that thereactive groups of the crosslinker have not yet reacted with thehaemostatic composition and are not hydrolyzed by water (or at least notin a significant amount which has negative consequences on thehemostatic functionality of the present compositions). This can beachieved by combining the crosslinked gelatin with the hydrophiliccrosslinker in a way which does not lead to reaction of the reactivegroups of the crosslinker with the hemostatic polymer or with water.Usually, this includes the omitting of aqueous conditions (or wetting),especially wetting without the presence of acidic conditions (ifcrosslinkers are not reactive under acidic conditions). This allows theprovision of reactive haemostatic materials.

Preferred ratios of the crosslinked gelatin to hydrophilic polymericcomponent in the hemostatic composition according to the presentinvention are from 0.1 to 50% (w/w), preferably from 5 to 40% (w/w).

Further components may be present in the hemostatic compositionaccording to the present invention. According to preferred embodiments,the hemostatic compositions according to the present invention mayfurther comprise a substance selected from the group consisting ofantifibrinolytic, procoagulant, platelet activator, antibiotic,vasoconstrictor, dye, growth factors, bone morphogenetic proteins andpain killers.

The present invention also refers to a finished final containercontaining the hemostatic composition according to the presentinvention. This finished container contains the hemostatic compositionaccording to the present invention in a sterile, storage-stable andmarketable form. The final container can be any container suitable forhousing (and storing) pharmaceutically administrable compounds.Syringes, vials, tubes, etc. can be used; however, providing thehemostatic compositions according to the present invention in a syringeis specifically preferred. Syringes have been a preferred administrationmeans for hemostatic compositions as disclosed in the prior art alsobecause of the handling advantages of syringes in medical practice. Thecompositions may then preferably be applied (after reconstitution) viaspecific needles of the syringe or via suitable catheters. Thereconstituted hemostatic compositions (which are preferablyreconstituted to form a hydrogel) may also be applied by various othermeans e.g. by a spatula, a brush, a spray, manually by pressure, or byany other conventional technique. Administration of the reconstitutedhemostatic composition to a patient by endoscopic (laparoscopic) meansis specifically preferred. Usually, the reconstituted hemostaticcompositions according to the present invention will be applied using asyringe or similar applicator capable of extruding the reconstitutedcomposition through an orifice, aperture, needle, tube, or other passageto form a bead, layer, or similar portion of material. Mechanicaldisruption of the compositions can be performed by extrusion through anorifice in the syringe or other applicator, typically having a size inthe range from 0.01 mm to 5.0 mm, preferably 0.5 mm to 2.5 mm.Preferably, however, the hemostatic composition will be initiallyprepared from a dry form having a desired particle size (which uponreconstitution, especially by hydration, yields subunits of therequisite size (e.g. hydrogel subunits)) or will be partially orentirely mechanically disrupted to the requisite size prior to a finalextrusion or other application step. It is, of course evident, thatthese mechanical components have to be provided in sterile form (insideand outside) in order to fulfill safety requirements for human use.

The hemostatic composition according to the present invention ispreferably applied in its pasty form to a patient from a container asdescribed in Example 1 with an extrusion force of 40 N or lower, such aslower 30N or lower 20N, preferably in a range of 15 to 30 N.

Another aspect of the invention concerns a method for providing aready-to-use hemostatic composition comprising contacting a hemostaticcomposition according to the present invention.

The invention is further described in the examples below and the drawingfigures, yet without being restricted thereto.

FIG. 1 shows the mean extrusion force of glutaraldehyde crosslinkedgelatin pastes containing 17.5% (w/w) crosslinked gelatin with variousconcentrations of human serum albumin in the thrombin component(extrusion force needed to push product out of syringe at compressionspeed 250 mm/min, calculated at 35 mm distance; all products incubatedfor 30 minutes at room temperature, quick re-swooshing shortly beforeextrusion force measurement). The x-axis shows the human serum albuminconcentration in the thrombin component in [g/l], the y-axis shows themean extrusion force in [N].

FIG. 2 shows the consistency of crosslinked gelatin pastes containing17.5% (w/w) crosslinked gelatin depending on the concentration of humanserum albumin.

FIG. 3 shows the mean extrusion force of genipin crosslinked gelatinpastes containing 17.5% (w/w) gelatin with various concentrations ofhuman serum albumin in the thrombin component. The x-axis shows thehuman serum albumin concentration in the thrombin component in [g/l],the y-axis shows the mean extrusion force in [N].

FIG. 4 shows evaluation of bleeding severity post test articleapplication and approximation.

FIGS. 5 to 8 show the hemostatic efficacy in Porcine Liver Punch-BiopsyModel of different preparations. The x-axis shows the time afterapplication in [seconds], the y-axis shows percent of hemostatic success(defined as “no bleeding” in FIG. 5 and as “no bleeding” or “ooze” inFIG. 6).

In FIGS. 5 and 6 the symbols mean:

glutaraldehyde crosslinked gelatin with 50 g/l human serum albumin inthe thrombin solution (n=8)

glutaraldehyde crosslinked gelatin with 75 g/l human serum albumin inthe thrombin solution (n=8)

In FIGS. 7 and 8 the symbols mean:

≈17.5% (w/w) glutaraldehyde crosslinked gelatin

≈14.5% (w/w) glutaraldehyde crosslinked gelatin

≈17.5% (w/w) glutaraldehyde crosslinked gelatin plus 2.5% PEG10.000 inthrombin solution

EXAMPLES Example 1 Determination of Extrusion Force (EF)

An Instron model 5544 mechanical tester equipped with a 100 N load celloperating at a cross-beam speed of 250 mm/min was used to measureextrusion forces needed to extrude the product from a syringe. Thenecessary extrusion forces were measured during the complete cross-beamdisplacement (34 mm deflection) which corresponds to a distance asyringe plunger moves in order to extrude almost the entire product outof the syringe. From these forces the mean extrusion forces werecalculated as follows:

$\frac{{Total}\mspace{14mu} {Energy}\mspace{14mu} ({mJ})}{{{Max}.\mspace{14mu} {Deflection}}\mspace{14mu} ({mm})} = {{Mean}\mspace{14mu} {Force}\mspace{14mu} (N)}$

Samples for this test were prepared as follows: A 5 ml standard syringe(with a cylindric body having an inner diameter of 12.2 mm) with a maleluer lock system (the inner nozzle lumen diameter where the adapter isattached measures 2.54 mm) is filled with 0.704 g dry mass of the solidsample (approx. 0.8 g taking the residual moisture of approx. 12% intoaccount). As a diluent 3.2 ml of a thrombin solution containing 500IU/ml thrombin in 40 mM calcium chloride and either 0, 5, 15, 25, 50 or75 mg/ml human serum albumin was used. The diluent and the solidcomponent were mixed by connecting the syringe holding the diluent (astandard 5 ml syringe with a female luer lock system) and the syringeholding the dry component and pushing the contents back and forth atleast 10 times (this mixing technique is called “swooshing”). Thereafterthe sample was incubated for 30 min at room temperature beforemeasurement. After incubation each sample was “re-swooshed” two timesand the syringe holding the product (the syringe that previously heldthe dry component as mentioned above) was connected to a malleableapplicator (female luer connector system, inner tube diameter of 2.29 mmholding two wires and having a total length of 141.5 mm). The syringewas assembled to the applicator and placed into the Instron set up andthe test was started.

The syringes and the applicator were commercially available as parts ofthe Floseal Hemostatic Matrix product from Baxter.

The results for a glutaraldehyde crosslinked gelatin as in Floseal aredepicted in FIG. 1 and those for a genipin crosslinked gelatin asdescribed below are depicted in FIG. 3 and also shown as correspondingTable 1 and Table 2.

The consistency of crosslinked gelatin pastes containing 17.5% (w/w)crosslinked gelatin depending on the concentration of albumin is shownin FIG. 2 (with 0, 25, 50 and 75 g/l human serum albumin provided in thethrombin component).

TABLE 1 c(albumin) [g/l] in the extrusion force std thrombin component[N] dev 0 40 2.4 5 38 1.5 15 30 2.6 25 25 2.2 50 19 1.5 60 19 1.0

TABLE 2 c(albumin) g/l in the extrusion force std thrombin component [N]dev 0 54 1.9 15 29 3.6 50 17 2.2

Preparation of Genipin Crosslinked Gelatin

Bovine derived collagen was processed via alkaline treatment andsubsequently rinsed with deionized process water (DIW) to removeresidual salts. Gelatin was extracted by heat treatment and dried insheets. The sheets were ground to a powder that was to be processedusing genipin as a crosslinking agent.

1 kg of gelatin granules were added to 201 of a 10 mM genipin solutionin DIW. The reaction was performed at neutral pH (7.2) in a jacketedtemperature controlled tank at 23° C. Mixing was carried out for 6 hoursand the solution was drained off, retaining the solids within a mesh,and rinsed through with DIW to wash out remaining genipin. The materialwas re-suspended in a 5% H₂O₂ solution for 20 hours. The material wasrinsed through with DIW to remove the H₂O₂. The solids were pre-dried onfilter paper under vacuum and then oven dried for 2.5 days. The driedmatrix was ground to a powder and filled into individual plasticsyringes before exposure to gamma irradiation.

Example 2 Determination of Hemostatic Efficiency Materials and MethodsAnimal Model

For this model, a midline laparotomy is performed, followed byelectrocautery to stop the bleeding from the surgical incision. Theliver is exposed and a lobe is isolated. A 10 mm diameter punch biopsyis used to create a series of 2, non-full thickness lesions,approximately 5 mm deep, with the core tissue removed. A pre-treatmentassessment is made on the lesion which includes collecting the bloodflowing from each lesion for 10 sec. with pre-weighed gauze.

Test articles are randomized and presented to the surgeon who is blindedto the sample treatment. Approximately 1.0 ml of the assigned testarticle is topically applied to a lesion. Saline moistened gauze is usedto help approximate the test articles to their designated lesions, andthe timer is started. The saline moistened approximation gauze isremoved after 30 seconds.

The degree of bleeding is assessed at 30, 60, 90, 120, 300, and 600 sec.after the test articles are applied to their designated lesions as perthe depictions in FIG. 3 (Bleeding score: 0: no bleeding (productsaturated with blood); 1: ooze (blood out of product but no blood drop);2: very mild (blood drop on the product); 3: mild (blood drop streamsdown); 4: moderate (small amount of blood streams down); 5: severe(large amount of blood streams down)).

Product saturated with blood, but without active bleeding is scored as a“0” (zero). Saline is used to irrigate the excess test articles awayfrom the lesions after the 300 sec. assessment. The procedure isrepeated and performed in multiple liver lobes. A single surgeoncreates, treats, and performs the observation assessments.

Test Article Synthesis

Test articles for the in vivo evaluation in the porcine-liver model weremade by preparing pastes of crosslinked gelatin (in concentrations of14.5% and 17.5% with 25 or 50 g/l human serum albumin in the thrombinsolution (with or without additional 2.5% PEG)).

The results are depicted in FIGS. 5 to 8 showing improved performancewith 17.5% gelatin and less effectiveness in the presence ofplasticizers (PEG).

Example 3

Gelatin samples were formulated per the package insert for Floseal witha couple key exceptions. First, sodium chloride was used instead ofcalcium chloride and the gelatin was formulated at 125% solids insteadof 100%. The gelatin/thrombin formulations were allowed to stand for 25minutes and then 1 ml of the preparation was discarded. The other 1 mlof material was applied to the topical hemostasis system (THS). The THSapparatus was previously primed with platelet poor plasma.

The THS is an apparatus designed to simulate a bleeding wound. Theartificial wound is a cylindrical hole in a silicone substrate. Thesurface of the silicone cylinder was coated with a layer of fibrinogen.A syringe pump expelled the clotting fluid (whole blood, plasma, etc.)in this case platelet poor plasma, while the back pressure was recorded.In this experiment the plasma was flowed at a fixed rate of 0.25 ml/minthrough a small hole at the bottom center of the cylindrical wound. Theexcess plasma was soaked up with gauze immediately prior to applicationof the hemostatic matrix. As the plasma continued to flow, 1 ml of thehemostatic matrix was applied to the cylindrical wound. This wasimmediately covered with wet gauze and a fixed pressure was applied.After 30 seconds the weight was removed and the plasma continued to flowfor 8-10 minutes, at which point the flow was stopped and the clot setaside in a humidity chamber where it stayed for more than 2 hours. Atthe end of the two hours, the clot was mounted onto a vibratome at 8°C., where approximately 500 μm thick slabs were sectioned from the clot.These sections were immersed into a PBS buffer. The slabs were stored ina 5° C. refrigerator when not in use. The slab was placed onto acoverslip and imaged with a Nikon A1R confocal microscope running theNIS-Elements Advanced Research v3.22.00 Build 710 software. To collectmicrographs, a plan fluor 10× objective was used with laser excitationlight at 488 nm and an emission collection window from 500-550 nm. Atransmitted light image was simultaneously collected using a transmittedlight detector. With these imaging parameters, automated stitchingperformed by the software was used to generate macroscopic maps ofsamples. Smaller areas of the samples were also characterized bycollecting 3D z-stacks of images with an optical slice thickness of5.125 μm. The composite confocal map was used to identify the gelatingranules that are located at the surface, and which were sectioned. Thiswas important for positioning of the elasticity measurement in theatomic force microscope (AFM). The clot slab was mounted in a VeecoMultimode AFM. The multimode was equipped with a Nanoscope V controllerand a JV piezoelectric scanner. The force measurements were made with aNovascan AFM cantiever which supported a 4.5 μm polystyrene sphere. Thecantilever's force constant was determined to be 0.779 N/m by thethermal tune method. The cantilever was positioned above the center ofthe gelatin granule, and then a 16×16 array of force measurements weremade. Each force curve involved moving the gelatin granule up intocontact with the polystyrene sphere, and continuing to move the granuleup until the cantilever deflection reached a preset trigger value of 2volts, at which point the gelatin was retracted a distance of 1.00micron from the trigger location.

Discussion

The fluorescence data shows that the glutaraldehyde crosslinked gelatinis not uniformly crosslinked. Instead, the crosslinking density seemshigher around the edges of the granules, with the central portion of thegranule being significantly less crosslinked than the edges. Incontrast, the genipin crosslinked gelatin appears uniformly(homogeneously) crosslinked throughout the granules. There are nosubstantial edge effects to the fluorescence intensity. The fluorescenceintensity of the genipin and glutaraldehyde crosslinked materials cannotbe directly compared, because of the potential fluorescence differencesattributed to the crosslinkers themselves. However, the AFM measuredelastic modulus measurement show that the genipin crosslinked gelatin isstiffer than the glutaraldehyde crosslinked gelatin, which appears to besofter (more flexible).

1. A hemostatic composition comprising crosslinked gelatin inparticulate form suitable for use in hemostasis, wherein the compositionis present in paste form containing 15.0 to 19.5% (w/w) crosslinkedgelatin, and wherein the composition comprises an extrusion enhancer. 2.The hemostatic composition according to claim 1, wherein the extrusionenhancer is albumin in an amount of between 0.5 to 5.0% (w/w).
 3. Thehemostatic composition according to claim 1, wherein the crosslinkedgelatin is glutaraldehyde-crosslinked gelatin or genipin-crosslinkedgelatin.
 4. The hemostatic composition according to claim 1, wherein thecrosslinked gelatin is type B gelatin.
 5. The hemostatic compositionaccording to claim 1, wherein the crosslinked gelatin is present asgranular material.
 6. The hemostatic composition according to claim 1,wherein the crosslinked gelatin has a particle size of 100 to 1000 μm.7. The hemostatic composition according to claim 1, wherein thecomposition contains thrombin.
 8. The hemostatic composition to claim 1for use in the treatment of an injury selected from the group consistingof a wound, a hemorrhage, a damaged tissue, a bleeding tissue, and abone defect.
 9. A method of treating an injury selected from the groupconsisting of a wound, a hemorrhage, a damaged tissue, and a bleedingtissue, comprising administering a hemostatic composition according toclaim 1 to the injury.
 10. A kit for making a flowable paste ofcrosslinked gelatin for the treatment of an injury selected from thegroup consisting of a wound, a hemorrhage, a damaged tissue, and ableeding tissue, comprising: a) a dry hemostatic composition comprisingcrosslinked gelatin in particulate form to be reconstituted to aflowable paste containing 15.0 to 19.5% (w/w) crosslinked gelatin; andb) a pharmaceutically acceptable diluent for reconstitution of thehemostatic composition, wherein either the composition or the diluentcomprises albumin in an amount which leads to an albumin concentrationin the reconstituted paste of between 0.5 to 5.0% (w/w).
 11. The kitaccording to claim 10, wherein the pharmaceutically acceptable diluentcomprises a buffer or buffer system.
 12. The kit according to claim 10,wherein the pharmaceutically acceptable diluent comprises thrombin. 13.The kit according to claim 10, wherein the pharmaceutically acceptablediluent contains a substance selected from the group consisting of NaCl,CaCl₂, and sodium acetate.
 14. A method for providing a ready to useform of a hemostatic composition according to claim 1, wherein thehemostatic composition is provided in a first syringe and a diluent forreconstitution is provided in a second syringe, the first and the secondsyringe are connected to each other, and the fluid is brought into thefirst syringe to produce a flowable form of the hemostatic composition.15. The method according to claim 14, wherein the flowable form of thehemostatic composition contains particles that are more than 50% (w/w)with a size of 100 to 1000 μm.
 16. The hemostatic composition accordingto claim 1, wherein the composition contains 16.0 to 19.5% (w/w)crosslinked gelatin.
 17. The hemostatic composition according to claim1, wherein the composition contains 16.5 to 19.5% (w/w) crosslinkedgelatin.
 18. The hemostatic composition according to claim 1, whereinthe composition contains 16.5 to 19.0% (w/w) crosslinked gelatin. 19.The hemostatic composition according to claim 1, wherein the compositioncontains 17.0 to 18.5% (w/w) crosslinked gelatin.
 20. The hemostaticcomposition according to claim 1, wherein the composition contains 16.5to 17.5% (w/w) crosslinked gelatin.